The detection of biomedically significant molecules with high-sensitivity nanoscale optical sensors has been the focus of major development efforts by many research groups worldwide (Fan et al., Anal. Chim. Acta 620:8-26 (2008)). Novel structures resulting from these efforts, including ring- and whispering-gallery resonators (Chao et al., Appl. Phys. Lett. 83:1527-1529 (2003); Armani et al., Science 317:783-787 (2007); Barrios et al., Opt. Lett. 32:3080-3082 (2007)), waveguides (Heideman et al., Sens. Actuators 10:209-217 (1993); Goddard et al., Analyst 119:583-588 (1994); Salamon et al., Biophys. J. 80:1557-1567 (2001)), and photonic crystals (Vollmer et al., Appl. Phys. Lett. 80:4057-4059 (2002); Krioukov et al., Opt. Lett. 27:1504-1506 (2002)), operate by resolving minute changes in refractive index that occur when a target molecule or virus interacts with the device. While all of these devices have remarkable theoretical sensitivities, their observed limits of detection (“LoD”) under real-world conditions are often unsatisfactory (Fan et al., Anal. Chim. Acta 620:8-26 (2008); Sheehan et al., Nano Lett. 5:803-807 (2005).
The LoD of a biosensor is dependent not only on the sensitivity of the transduction mechanism, but also on the biomolecular thermodynamics of the immobilized probe and the target analyte in solution (Lambeck, Meas. Sci. Technol. 17:R93-R116 (2006); Kusnezow et al., Mol. Cell. Proteomics 5:1681-1696 (2006)). In addition to presenting unique challenges for analyte mass transport, nanoscale sensors require careful functionalization with capture molecules (for example, antibodies) since the active sensing region is orders of magnitude smaller than the overall device. If the placement of capture molecules (probes) onto the surface is indiscriminate and both the sensing and non-sensing regions are functionalized (Sapsford et al., Anal. Chem. 73:5518-5524 (2001); Choi et al., Anal. Biochem. 405:1-10 (2010)), the target loss to the non-sensing regions may become substantial enough to disturb the bulk concentration of target. This can lead to a lower fraction of material being bound to the sensing area, and a higher (worse) LoD (Ekins et al., Clin. Chem. 37:1955-1967 (1991); Ekins, Clin. Chem. 44:2015-2030 (1998); Parpia et al., Anal. Biochem. 401:1-(2010)). Conventional passivation techniques (Taylor et al., Nucleic Acids Res. 31:e87 (2003)) involving incubation with proteins (e.g. bovine serum albumin) or synthetic blocking chemicals cannot be used to avoid this issue, since they would result in equal application to the non-sensing and sensing areas of nanoscale devices. A common top-down approach to this problem has been to shrink the size of the probe droplet in manufacturing to closely overlay only the active sensing region (McKendry et al., Proc. Natl. Acad. Sci. U.S.A. 99:9783-9788 (2002); Lee et al., Nano Lett. 4:1869-1872 (2004)). However, there are considerable challenges with alignment and uniform dispensing on such a small scale. Others have exploited material differences within a nanoscale biosensor. For example, Fuez et al. showed material-selective surface chemistry that selectively bound a blocking agent to inactive titanium dioxide surfaces of a plasmonic nanostructure leaving the gold sensing region to bind biomolecules (Feuz et al., ACS Nano 4:2167-2177 (2010)). Since incorporation of different materials into the device is not always feasible, alternative strategies are clearly needed.
The devices and methods disclosed herein are directed to overcoming these and other deficiencies in the art.